Communication Twisted Leaf : A Novel Indigenous New World Monopartite Infecting Tomato in Venezuela

Gustavo Romay 1,*, Francis Geraud-Pouey 2, Dorys T. Chirinos 3, Mathieu Mahillon 1, Annika Gillis 4,† , Jacques Mahillon 4 and Claude Bragard 1,*

1 UCLouvain, Earth and Life Institute, Applied Microbiology-Phytopathology, Croix du Sud 2-L07.05.03, 1348 Louvain-la-Neuve, Belgium; [email protected] 2 La Universidad del Zulia (LUZ), Unidad Técnica Fitosanitaria, Maracaibo 4005, Estado Zulia, Venezuela; [email protected] 3 Facultad de Ingeniería Agronómica, Universidad Técnica de Manabí, Manabí 130105, Ecuador; [email protected] 4 UCLouvain, Earth and Life Institute, Applied Microbiology-Laboratory of Food and Environmental Microbiology, Croix du Sud 2-L7.05.12, 1348 Louvain-la-Neuve, Belgium; [email protected] (A.G.); [email protected] (J.M.) * Correspondence: [email protected] (G.R.); [email protected] (C.B.) † Present address: Section of Microbiology and Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London SW72AZ, UK.

 Received: 21 February 2019; Accepted: 2 April 2019; Published: 4 April 2019 

Abstract: are one of the major groups of plant viruses with an important economic impact on crop production in tropical and subtropical regions. The global spread of its polyphagous vector, the whitefly Bemisia tabaci, has contributed to the emergence and diversification of species within this . In this study, we found a putative novel begomovirus infecting tomato plants in Venezuela without a cognate DNA-B component. This begomovirus was genetically characterized and compared with related species. Furthermore, its infectivity was demonstrated by agroinoculation of infectious clones in tomato (Solanum lycopersicum) and Nicotiana benthamiana plants. The name Tomato twisted leaf virus (ToTLV) is proposed. ToTLV showed the typical organization of the DNA-A component of New World bipartite begomoviruses. However, the single DNA component of ToTLV was able to develop systemic infection in tomato and N. benthamiana plants, suggesting a monopartite nature of its genome. Interestingly, an additional open reading frame ORF was observed in ToTLV encompassing the intergenic region and the coat protein gene, which is not present in other closely related begomoviruses. A putative transcript from this region was amplified by strand-specific reverse transcription-PCR. Along with recent studies, our results showed that the diversity of monopartite begomoviruses from the New World is greater than previously thought.

Keywords: begomovirus evolution; ; Solanum lycopersicum; tomato crop

1. Introduction Over the course of the last few decades, members of the genus Begomovirus have remained serious threats to tomato production in tropical and subtropical regions across the world. Begomoviruses represent the largest group of plant viruses and belong to the family Geminiviridae along with members of eight different genera: Becurtovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, , Topocuvirus, and [1]. One of the most important factors mediating the ever-increasing emergence and dissemination of begomoviruses is the global spread of its vector, the whitefly Bemisia

Viruses 2019, 11, 327; doi:10.3390/v11040327 www.mdpi.com/journal/viruses Viruses 2019, 11, 327 2 of 11 tabaci [2]. The genome of begomoviruses has one or two circular single-stranded DNA components, referred to as DNA-A and DNA-B [3]. The DNA-A of bipartite begomoviruses is homologous to the genome of monopartite begomoviruses and encodes the protein (open reading frame (ORF) AV1/V1), the AV2/V2 protein (ORF AV2/V2), the replication-associated protein (ORF AC1/C1), the transcriptional activator protein (ORF AC2/C2), the replication enhancer protein (ORF AC3/C3), and the AC4/C4 protein (ORF AC4/C4), while the DNA-B encodes the nuclear shuttle protein (ORF BV1) and the movement protein (ORF BC1) [1]. For taxonomical purposes, the full-length sequence of DNA-A, or its homologous DNA of monopartite begomoviruses, is used to classify and distinguish species into their begomovirus group considering 91% of nucleotide identity as the demarcation threshold [3]. Begomoviruses can be separated in two geographically distant groups, the New World (NW) group and the Old World (OW) group. In general, NW begomoviruses have a bipartite genome, while the majority of OW begomoviruses (ca. 85%) display a monopartite genome [2]. Both DNA-A and DNA-B components of bipartite begomoviruses are required for systemic infection [3]: while DNA-A is responsible for replication and transcription functions, DNA-B is implicated in the viral movement [4]. Unlike bipartite begomoviruses, only the single DNA component of monopartite begomoviruses is essential to develop systemic infection [4]. Another difference between both geographic groups is the lack of the AV2 gene in the genome of NW begomoviruses [1]. Although NW begomoviruses had been thought to have only bipartite , a new tomato begomovirus containing a single DNA component was reported in Peru in 2011 [5]. This virus was named Tomato leaf deformation virus (ToLDeV) and its infectivity was demonstrated through development of several infectious clones [6,7]. In this study, a putative new begomovirus species infecting tomato plants was found in Venezuela containing a single DNA component. Further assays to fulfill Koch’s postulates were conducted with this novel virus and demonstrated the monopartite nature of its genome.

2. Materials and Methods

2.1. Sample Collection and Begomovirus Detection In July 2005, during a survey of begomoviruses carried out in Venezuela [8], leaf samples were collected in Zulia state from two tomato plants (samples 2005-07-22-01 and 2005-07-22-02; hereafter referred to as 331 and 332, respectively) exhibiting leaf deformation and stunting symptoms. Each sample was dried on a sealed receptacle containing Silicagel®. The dried samples were wrapped on tissue paper and stored in 2 mL tubes at −20 ◦C until use. Total DNA was extracted using a standard protocol [9] and PCR detection for begomoviruses was performed using the degenerate primer pairs PAL1v1978/PAR1c946 and PBLv2040/PCRc1 [10] and Taq DNA Polymerase master mix (New England BioLabs, USA). PCR conditions were as follows: 2 min at 94 ◦C, 35 cycles at 94, 55, and 68 ◦C for 30, 30, and 60 s, respectively, and a final extension of 5 min at 68 ◦C. PCR products were cloned in Escherichia coli competent cells using the pGem®-T Easy vector system (Promega, USA). At least five clones were sequenced for further BLAST analysis.

2.2. Cloning of Full-Length Viral Genomes Using tomato samples 331 and 332, a 1.1 kb fragment was cloned and subsequently sequenced. Since they shared 99.8% of identity, only sample 332 was considered for subsequent studies. DNA from this sample was used as a template to obtain the viral sequences by rolling circle amplification (RCA) using Φ29 DNA polymerase (TempliPhi, GE Healthcare, Munich, Germany). Restriction enzymes BamHI, EcoRI, HindIII, and PstI were used to follow a protocol of restriction fragment length polymorphism (RFLP) on RCA products (RFLP-RCA) according to Haible et al. [11]. The bands were excised from agarose gel and inserted into the appropriate restriction sites in pBluescript II SK (+) (La Jolla, Stratagene, CA, USA) for subsequent transformation of E. coli competent cells. Viruses 2019, 11, 327 3 of 11

The cloned products were sequenced at Macrogen Inc. (Amsterdam, Netherlands). A preliminary identification of viral sequences was based on a BLAST search and three different begomovirus DNA components were observed, suggesting a mixed infection. Subsequently, the three different full-length viral clones, referred to as isolates Be1.1B, Be6.6H, and B6.7H, were completely sequenced by primer walking and assembled using BioEdit v.7.1.9 [12]. The complete nucleotide sequence of clones Be6.6H, Be6.7H, and Be1.1B were deposited in GenBank under the accession numbers MK440292, MK440293, and MK440294, respectively.

2.3. Pairwise Identity, Phylogenetic, and Recombination Analyses Since the isolate Be6.6H did not show more than 90% of nucleotide identity with any begomovirus in GenBank database, it was considered for further genetic and biological studies. Isolate Be6.6H was aligned with sequences of related NW begomoviruses using MUSCLE [13] and pairwise sequence identities were estimated using the Sequence Demarcation Tool (SDT) v.1.2 [14]. The aligned sequences were analyzed to determine the optimal nucleotide substitution model and generate a maximum-likelihood phylogenetic tree (500 bootstrap replicates) using MEGA X v10.0.5 [15]. The phylogenetic tree was rooted with the OW begomovirus species African (ACMV). In order to identify potential recombination events in isolate Be6.6H, putative parental begomoviruses were downloaded from GenBank based on sequence analysis with the software SWeBLAST [16]. These viruses were aligned to perform recombination analyses using the methods BoostScan, Chimera, GENCOV, MaxChi, RDP, SiScan, and 3Seq implemented in RDP4 software v.4.31 [17]. Only recombination events detected by at least five out of seven methods were considered.

2.4. Construction of an Infectious Clone of Isolate Be6.6H for Agroinoculation A cognate DNA-B component of the isolate Be6.6H was found neither by PCR with begomovirus universal primers [10] nor by cloning of digested RCA products. In order to evaluate the infectivity of Be6.6H isolate, a multimeric clone was developed, as follows: the viral clone inserted in the HindIII site of pBluescript II SK (+), here referred to as pBe6.6H, was released with double digestion using enzymes BamHI and HindIII. This viral fragment was inserted into pCAMBIA1300 to generate a 0.9-mer clone containing the viral intergenic region. Then, the plasmid pBe6.6H was digested with HindIII to release the full-length viral DNA and then inserted into the plasmid pCAMBIA1300 containing the 0.9-mer clone of Be6.6H isolate. The generated 1.9-mer clone was named pBe6.6Hdi. The correct orientation of the HindIII-HindIII insert was confirmed by SacI restriction assays. Agrobacterium tumefaciens cells (strain C58C1) were transformed with plasmid pBe6.6Hdi by electroporation. PCR assays using specific primers, Be-2147F (50-ACGGCATTGGCGTCTTTGG-30) and Be-510R (50-TCGTCCATCCATATCTTGCC-30), were performed to confirm the presence of viral DNA in the bacteria. At least six single colonies of A. tumefaciens harboring pBe6.6Hdi were grown at 28 ◦C for 48 h in 5 mL of Luria–Bertani (LB) medium supplemented with kanamycin (50 mg/L) and rifampicin (25 mg/L). Then, 50 ml of LB containing antibiotics were inoculated with 5 ml of A. tumefaciens culture and grown at 28 ◦C for 16 h with shaking. The bacteria were harvested by centrifugation at 2000 g for 10 min. The pellets were resuspended in 30 mL of MMA solution (10 mM MgCl2, 10 mM MES (pH 5.6), 100 µM acetosyringone). Then, the pellets were centrifuged at 5000 g for 10 min and resuspended again in 30 mL of MMA solution. The bacterial suspensions were kept at room temperature with shaking for 4 h. The final optical density at 600 nm (OD600) was adjusted to 0.8. Tomato cv. Moneymaker and Nicotiana benthamiana plantlets were infiltrated on the lower surface of young fully expanded leaves using a 1 mL needleless syringe. Ten plants of tomato and N. benthamiana were inoculated with the viral clone. As negative control, 10 plants of both species were infiltrated with a mock (A. tumefaciens carrying the empty pCAMBIA1300 vector). Three replicates of this test were done. The viral infection was checked by PCR with primers Be-2147F and Be-510R at three weeks post inoculations (wpi). Viruses 2019, 11, 327 4 of 11

2.5. Genetic and Functional Analyses of a Sixth ORF in Be6.6H Isolate A sixth viral ORF was found in both isolates from tomato samples, starting at position 84 from the cleavage site “AC” of the invariant nonanucleotide TAATATT|AC at the origin of replication. The deduced amino acid (aa) sequence was subjected to a BLASTp search. In order to determine whether a transcript is produced from this ORF, total RNA was extracted from young leaf tissue of tomato plants positive to Be6.6H isolate using TRIzol reagent (Invitrogen, USA). RT-PCR assays were performed with primers Be-089F (50-GAGAGCGTCTGTGGAGCCT-30) and Be-363R (50-TGAACCTTACATGGGCCTTC-30) and Be-2147F/Be-363R using a OneTaq® One-Step RT-PCR Kit (New England BioLabs Inc., USA). Prior to RT-PCR assays, RNA extractions were treated with RQ1 RNase-Free DNase (Promega, USA) to prevent DNA viral contamination. RNA extractions, RT-PCR, and DNase treatments were performed following protocols suggested by the manufacturers.

3. Results

3.1. Sample Collection and Begomovirus Detection Begomovirus PCR assays confirmed the viral infection in tomato plants showing leaf deformation and stunting symptoms in Zulia estate, Venezuela. The PCR tests yielded expected fragment sizes (ca. 1.1 kb). The samples 331 and 332 showed a viral sequence sharing 99.8% nucleotide identity (NI) with each other, and 85% NI with the closest related begomovirus species Abutilon golden mosaic Yucatan virus (AbGMYV, GenBank No. KC430935). In addition, sample 332 contained another viral sequence with the highest identity (98%) with Tomato chlorotic leaf distortion virus (ToCLDV, GenBank No. JN241632), indicating a mixed infection in this sample. When using degenerate primers for DNA-B, only sample 332 was positive showing the expected fragment size (ca. 500 bp). This fragment was then cloned and at least 20 clones were sequenced. They all shared 95% NI with DNA-B of ToCLDV (GenBank No. JN241633).

3.2. Cloning of Full-Length Viral Genome The RFLP-RCA assays confirmed the mixed infection caused by begomoviruses in sample 332. After cloning of restricted fragments, three begomovirus DNA components were identified. The clone Be6.6H (2597 bp) exhibited the highest sequence identity (82%) with the DNA-A component of Rhynchosia golden mosaic Yucatan virus (GenBank No. EU021216), which is currently considered to be a strain of the species Cabbage leaf curl virus (CabLCV) [1]. The isolate Be6.6H showed the five typical ORFs found in the genome organization of DNA-A from NW bipartite begomoviruses (Figure1A). Interestingly, an additional ORF of 342 nucleotides, named ORF 6, was noticed encompassing the intergenic region (IR) and the V1 gene that codes for the coat protein (CP) (Figure1B). In OW begomovirus genome, the V2/AV2 gene is present in the same region as the ORF 6 (Figure1C). However, no homology was found between the V2 or AV2 protein and the deduced aa sequence from ORF 6 of Be6.6H. The clone Be6.7H (2623 bp) shared 99% NI with a ToCLDV DNA-A component (GenBank No. JN241632) isolated from chinense, while the clone Be1.1B (2608 bp) shared the highest sequence identity (94%) with a ToCLDV DNA-B component (GenBank No. HQ201953) infecting tomato [18]. As expected, clones Be6.7H and Be1.1B shared a 172 nt common region with 93.5% of identity, indicating that they are a cognate pair of the same ToCLDV isolate. Meanwhile, Be6.6H showed a low homology (ca. 77 % NI) of this genome region with clones Be6.7H and Be1.1B. Clone Be6.6H displayed two copies of iteron CTGGAGTC and two inverted copies (GACTCCAG) of this iteron upstream of the TATA box, while clones Be6.7H and Be1.1B only showed two copies of the same iteron sequence (TGTATTGG) (Figure2). The iteron sequences are required for viral replication and they are specific to each begomovirus species. Viruses 2019, 11, x FOR PEER REVIEW 4 of 11

2.5. Genetic and Functional Analyses of a Sixth ORF in Be6.6H Isolate A sixth viral ORF was found in both isolates from tomato samples, starting at position 84 from the cleavage site “AC” of the invariant nonanucleotide TAATATT|AC at the origin of replication. The deduced amino acid (aa) sequence was subjected to a BLASTp search. In order to determine whether a transcript is produced from this ORF, total RNA was extracted from young leaf tissue of tomato plants positive to Be6.6H isolate using TRIzol reagent (Invitrogen, USA). RT-PCR assays were performed with primers Be-089F (5′-GAGAGCGTCTGTGGAGCCT-3′) and Be-363R (5′-TGAACCTTACATGGGCCTTC-3′) and Be-2147F/Be-363R using a OneTaq® One-Step RT-PCR Kit (New England BioLabs Inc., USA). Prior to RT-PCR assays, RNA extractions were treated with RQ1 RNase-Free DNase (Promega, USA) to prevent DNA viral contamination. RNA extractions, RT-PCR, and DNase treatments were performed following protocols suggested by the manufacturers.

3. Results

3.1. Sample Collection and Begomovirus Detection Begomovirus PCR assays confirmed the viral infection in tomato plants showing leaf deformation and stunting symptoms in Zulia estate, Venezuela. The PCR tests yielded expected fragment sizes (ca. 1.1 kb). The samples 331 and 332 showed a viral sequence sharing 99.8% nucleotide identity (NI) with each other, and 85% NI with the closest related begomovirus species Abutilon golden mosaic Yucatan virus (AbGMYV, GenBank No. KC430935). In addition, sample 332 contained another viral sequence with the highest identity (98%) with Tomato chlorotic leaf distortion virus (ToCLDV, GenBank No. JN241632), indicating a mixed infection in this sample. When using degenerate primers for DNA-B, only sample 332 was positive showing the expected fragment size (ca. 500 bp). This fragment was then cloned and at least 20 clones were sequenced. They all shared 95% NI with DNA-B of ToCLDV (GenBank No. JN241633).

3.2. Cloning of Full-Length Viral Genome The RFLP-RCA assays confirmed the mixed infection caused by begomoviruses in sample 332. After cloning of restricted fragments, three begomovirus DNA components were identified. The clone Be6.6H (2597 bp) exhibited the highest sequence identity (82%) with the DNA-A component of Rhynchosia golden mosaic Yucatan virus (GenBank No. EU021216), which is currently considered to be a strain of the species Cabbage leaf curl virus (CabLCV) [1]. The isolate Be6.6H showed the five typical ORFs found in the genome organization of DNA-A from NW bipartite begomoviruses (Figure 1A). Interestingly, an additional ORF of 342 nucleotides, named ORF 6, was noticed encompassing the intergenic region (IR) and the V1 gene that codes for the coat protein (CP) (Figure 1B). In OW begomovirus genome, the V2/AV2 gene is present in the same region as the ORF 6 Viruses(Figure2019 1C)., 11 ,However, 327 no homology was found between the V2 or AV2 protein and the deduced5 of aa 11 sequence from ORF 6 of Be6.6H. Viruses 2019, 11, x FOR PEER REVIEW 5 of 11

Figure 1. Genome organization of begomoviruses. (A) Typical genome organization of the DNA-A component from New World begomoviruses. (B) Genome organization of the clone Be6.6H displaying a sixth ORF encompassing the intergenic region (IR) and the V1 gene encoding the coat protein. (C) Typical genome organization of a single DNA or DNA-A component from monopartite and bipartite begomoviruses of the Old World. The gray lines represent the genome size of single DNA or DNA-A component of begomoviruses (ca. 2600–2800 bp). The arrowheads indicate the sense 5ˊ–3ˊ to each gene.

The clone Be6.7H (2623 bp) shared 99% NI with a ToCLDV DNA-A component (GenBank No. JN241632) isolated from Capsicum chinense, while the clone Be1.1B (2608 bp) shared the highest sequenceFigure 1. identityGenome (94%) organization with a of ToCLDV begomoviruses. DNA- (BA )component Typical genome (GenBank organization No. HQ201953) of the DNA-A infecting tomatocomponent [18]. fromAs expected, New World clones begomoviruses. Be6.7H and (B) GenomeBe1.1B shared organization a 172 of nt the common clone Be6.6H region displaying with 93.5% of identity,a sixth ORFindicating encompassing that they the are intergenic a cognate region pair (IR) of andthe thesame V1 ToCLDV gene encoding isolate. the Meanwhile, coat protein. Be6.6H showed(C) Typical a low genome homology organization (ca. 77 % of NI) a single of this DNA genome or DNA-A region componentwith clones from Be6.7H monopartite and Be1.1B. and bipartiteClone begomoviruses Be6.6H displayed of the Old two World. copies The gray of linesiteron represent CTGGAGTC the genome and size oftwo single inverted DNA or copies 0 0 (GACTCCAG)DNA-A component of this of begomovirusesiteron upstream (ca. 2600–2800of the TATA bp). The box, arrowheads while clones indicate Be6.7H the sense and 5 –3Be1.1Bto only showedeach gene. two copies of the same iteron sequence (TGTATTGG) (Figure 2). The iteron sequences are required for viral replication and they are specific to each begomovirus species.

Figure 2. Iteron sequences found in the intergenic region of the clones Be6.6H, Be6.7H, and Be1.1B. CloneFigure Be6.6H 2. Iteron displayed sequences different found iterons in the as comparedintergenic toregion clones of Be6.7H the clon andes Be1.1B.Be6.6H, Sequences Be6.7H, and in lightBe1.1B. greyClone boxes Be6.6H represent displayed iteron sequences. different Rightiterons arrows as compar indicateed forwardto clones repeats Be6.7H of an iterons,d Be1.1B. and leftSequences arrows in indicatelight grey inverted boxes repeats represent of the iteron iterons. sequences. Right arrows indicate forward repeats of iterons, and left arrows indicate inverted repeats of the iterons. 3.3. Pairwise, Phylogenetic, and Recombination Analyses 3.3.The Pairwise, full-length Phylogenetic, sequence and of isolateRecombination Be6.6H wasAnalyses compared with begomovirus sequences available in the GenBankThe full-length dataset usingsequence Blastn. of Tableisolate1 showsBe6.6H the was six mostcompared related with begomoviruses begomovirus to isolatesequences Be6.6H.available According in the GenBank to SDT pairwise dataset scores, using thisBlastn. isolate Tabl showede 1 shows the the greatest six most similarities, related begomoviruses 82.4 and 82.1%, to to anisolate isolate Be6.6H. of Euphorbia According mosaic to SDT virus pairwise(EuMV) fromscores, Cuba this (GenBankisolate showed No.KU165788) the greatest and similarities, a CabLCV 82.4 isolateand (GenBank82.1%, to an No. isolate EU021216) of Euphorbia from mosaic , virus respectively. (EuMV) from Begomoviruses Cuba (GenBank sharing No. KU165788) DNA-A NI and < a 91%CabLCV are recognized isolate (GenBank as different No. species EU021216) [1]. Therefore, from Mexico, isolate Be6.6Hrespectively. represents Begomoviruses a putative novelsharing begomovirusDNA-A NI species < 91% and are therecognized name Tomato as different twisted specie leaf viruss [1]. (ToTLV) Therefore, is proposed isolate forBe6.6H this virus.represents a putative novel begomovirus species and the name Tomato twisted leaf virus (ToTLV) is proposed Table 1. Percent nucleotide sequence identity between Tomato twisted leaf virus (ToLTV) genome and for this virus. closely related begomoviruses.

1 BegomovirusTable 1. Percent Species nucleotide ToTLVsequence identity EuMV between CabLCV Tomato ToMYLCV twisted leaf BLCrVvirus (ToLTV) JacYVV genome and closely related begomoviruses. ToTLV (Venezuela) 100.0 1BegomovirusEuMV (Cuba) species 82.4 100.0 ToTLV EuMV CabLCV ToMYLCV BLCrV JacYVV CabLCV (Mexico) 82.1 82.5 100.0 ToMYLCV (Venezuela) 81.7 88.1 82.9 100.0 ToTLV (Venezuela) 100.0 BLCrV (Colombia) 81.2 82.6 83.3 84.1 100.0 EuMV (Cuba) 82.4 100.0 JacYVV (Venezuela) 81.1 81.0 79.3 79.8 78.9 100.0 CabLCVEuYMV (Mexico) (Brazil) 80.7 82.1 82.3 82.5 80.7 100.0 84.6 80.5 77.3 ToMYLCV (Venezuela) 81.7 88.1 82.9 100.0 1 Acronyms refer to Tomato twisted leaf virus (ToTLV; MK440292), Euphorbia mosaic virus (EuMV; KU165788), CabbageBLCrV leaf curl (Colombia) virus (CabLCV; EU021216), 81.2 Tomato mild 82.6 yellow leaf curl 83.3 Aragua virus (ToMYLCV; 84.1 JN368145), 100.0 leaf crumpleJacYVV virus (Venezuela)(BLCrV; KX857725), Jacquemontia 81.1 yellow 81.0 vein virus (JacYVV; 79.3 KY617094), 79.8 and Euphorbia 78.9 yellow mosaic 100.0 virus (EuYMV;EuYMV KY559488).(Brazil) Country of 80.7 origin to each82.3 viral isolate is80.7 in brackets. 84.6 80.5 77.3 1 Acronyms refer to Tomato twisted leaf virus (ToTLV; MK440292), Euphorbia mosaic virus (EuMV; KU165788), Cabbage leaf curl virus (CabLCV; EU021216), Tomato mild yellow leaf curl Aragua virus (ToMYLCV; JN368145), Bean leaf crumple virus (BLCrV; KX857725), Jacquemontia yellow vein virus

Viruses 2019, 11, x FOR PEER REVIEW 6 of 11 Viruses 2019, 11, 327 6 of 11 (JacYVV; KY617094), and Euphorbia yellow mosaic virus (EuYMV; KY559488). Country of origin to each viral isolate is in brackets. The phylogenetic analysis indicated that ToTLV belongs to the Squash leaf curl virus (SCLV) clade The phylogenetic analysis indicated that ToTLV belongs to the Squash leaf curl virus (SCLV) (Figure3), a genetic group of begomoviruses widely distributed in the Americas [ 19], although forming clade (Figure 3), a genetic group of begomoviruses widely distributed in the Americas [19], although a relative independent branch within this clade. forming a relative independent branch within this clade.

Figure 3. Phylogenetic relationships between the full-length DNA sequence of the novel begomovirus Figure 3. Phylogenetic relationships between the full-length DNA sequence of the novel Tomato twisted leaf virus (highlighted in bold) and those of related begomoviruses. The phylogenetic begomovirus Tomato twisted leaf virus (highlighted in bold) and those of related begomoviruses. tree is based on the Maximum likelihood method using GTR+G as a nucleotide substitution model. The phylogenetic tree is based on the Maximum likelihood method using GTR+G as a nucleotide Bootstrap values (500 iterations) above 50% are indicated for each node. African substitution model. Bootstrap values (500 iterations) above 50% are indicated for each node. African (ACMV) was used as an outgroup. GenBank accession numbers are indicated in brackets. cassava mosaic virus (ACMV) was used as an outgroup. GenBank accession numbers are indicated in Recombinationbrackets. analysis predicted the occurrence of a recombination event in the genome of ToTLV by seven methods implemented in RDP4 software. SiScan showed the lowest p value (p = 7.195Recombination× 10−25). Recombination analysis predicted breakpoints the occurrence were detected of a recombination at nucleotide positionsevent in 1942the genome (ORF C1) of andToTLV 24 (intergenic by seven region)methods of implemented ToTLV, suggesting in RDP4 that thissoftware. virus hasSiScan a recombinant showed the nature. lowestTomato p value yellow (p = −25 mottle7.195 virus× 10 (ToYMoV,). Recombination GenBank breakpoints No. KY064015) were and detectedJacquemontia at nucleotide yellow veinpositions virus (JacYVV,1942 (ORF GenBank C1) and No.24 (intergenic KY617094) region) were identified of ToTLV, as putativesuggesting major that and this minor virus parents,has a recombinant respectively. nature. ToYMoV Tomato has yellow been mottle virus (ToYMoV, GenBank No. KY064015) and Jacquemontia yellow vein virus (JacYVV, GenBank

Viruses 2019, 11, x FOR PEER REVIEW 7 of 11 Viruses 2019, 11, 327 7 of 11 No. KY617094) were identified as putative major and minor parents, respectively. ToYMoV has been reported infecting tomato in Costa Rica [20], while JacYVV was described in Venezuela infecting Jacquemontiareported infecting tamnifolia tomato, a wild in plant Costa belonging Rica [20], to while the family JacYVV Convolvulaceae was described [21]. in Venezuela infecting Jacquemontia tamnifolia, a wild plant belonging to the family Convolvulaceae [21]. 3.4. Infectivity of Clone Be6.6H in Tomato and N. Benthamiana Plants 3.4. Infectivity of Clone Be6.6H in Tomato and N. Benthamiana Plants A. tumefaciens cells, containing the 1.9-mer clone of ToTLV, were inoculated in tomato and N. A. tumefaciens cells, containing the 1.9-mer clone of ToTLV, were inoculated in tomato and N. benthamiana plants. After three wpi, the virus was detected by PCR in young leaves of both plants, benthamiana plants. After three wpi, the virus was detected by PCR in young leaves of both plants, indicating the systemic infection of ToTLV isolate Be6.6H. At three wpi, the transmission rate was indicating the systemic infection of ToTLV isolate Be6.6H. At three wpi, the transmission rate was 70% 70% (7, 6, and 8 out of 10 plants used for each replicate, respectively). After four wpi, (7, 6, and 8 out of 10 plants used for each replicate, respectively). After four wpi, ToTLV-infected tomato ToTLV-infected tomato plants showed a reduced growth, as compared with mock-inoculated plants plants showed a reduced growth, as compared with mock-inoculated plants (Figure4A). The tomato (Figure 4A). The tomato plants also displayed twisted and curled leaves (Figure 4B). Furthermore, plants also displayed twisted and curled leaves (Figure4B). Furthermore, severe symptoms consisting severe symptoms consisting of leaf deformation and a drastic reduction in the size of the new of leaf deformation and a drastic reduction in the size of the new emergent leaves were observed at emergent leaves were observed at eight wpi (Figure 4C). In N. benthamiana plants, mosaic symptoms eight wpi (Figure4C). In N. benthamiana plants, mosaic symptoms associated with ToTLV were also associated with ToTLV were also observed after four wpi (Figure S1) and the transmission rate was observed after four wpi (Figure S1) and the transmission rate was 100%. 100%.

FigureFigure 4. 4. (A(A)) Symptoms Symptoms associated associated with with ToTLV ToTLV infection infection in in tomato tomato plants plants (cv. (cv. Moneymayker) Moneymayker) after after agroinoculation.agroinoculation. Mock-inoculated Mock-inoculated (left) (left) and and ToTLV- ToTLV-inoculatedinoculated (right) (right) plants plants at at four four weeks weeks post post inoculationinoculation (wpi). (wpi). (B) (B )Twisted Twisted leaf leaf symptoms symptoms observed observed in in ToTLV-inoculated ToTLV-inoculated plants. (C) (C )Severe Severe symptomssymptoms (e.g., (e.g., size size reduction reduction of of young young leaves, leaves, chlorosis, chlorosis, and and leaf leaf deformation) deformation) observed observed in in tomato tomato plantsplants after after eight eight wpi. wpi. 3.5. Genetic Analysis of a Putative Novel Sixth ORF in Be6.6H Isolate 3.5. Genetic Analysis of a Putative Novel Sixth ORF in Be6.6H Isolate The genome analysis revealed a sixth viral ORF in ToTLV encompassing the intergenic and the The genome analysis revealed a sixth viral ORF in ToTLV encompassing the intergenic and the CP coding region. BLASTp analysis did not show significant similarity to any protein in public CP coding region. BLASTp analysis did not show significant similarity to any protein in public databases. RT-PCR assays from ToTLV-infected tomato plants yielded an amplicon of ca. 300 nt when databases. RT-PCR assays from ToTLV-infected tomato plants yielded an amplicon of ca. 300 nt using primers Be-089F and Be-363R (Figure5A), indicating that this ORF is potentially transcribed. when using primers Be-089F and Be-363R (Figure 5A), indicating that this ORF is potentially The amplified fragment covered an internal region from position 2 to 100 of the putative protein transcribed. The amplified fragment covered an internal region from position 2 to 100 of the putative coded by ORF 6. In order to exclude false positive results due to viral DNA contamination in RNA protein coded by ORF 6. In order to exclude false positive results due to viral DNA contamination in extractions, RT-PCR assays were performed using primer Be-2147F targeting the AC1 gene and primer RNA extractions, RT-PCR assays were performed using primer Be-2147F targeting the AC1 gene Be-363R targeting ORF 6 (Figure5B). Both primers were also used for PCR using DNA extractions and primer Be-363R targeting ORF 6 (Figure 5B). Both primers were also used for PCR using DNA from the same leaf tissues. RT-PCR assays did not yield any amplification products, while the PCR extractions from the same leaf tissues. RT-PCR assays did not yield any amplification products, tests yielded the expected 850 bp fragment using the same primer pairs (Figure5A). Although RT-PCR while the PCR tests yielded the expected 850 bp fragment using the same primer pairs (Figure 5A). assays suggest that the ORF 6 is transcribed, 50 and 30 Rapid amplification of cDNA ends (RACE) Although RT-PCR assays suggest that the ORF 6 is transcribed, 5′ and 3′ Rapid amplification of assays and mutational analysis are required to confirm these results. cDNA ends (RACE) assays and mutational analysis are required to confirm these results.

Viruses 2019, 11, 327 8 of 11 Viruses 2019, 11, x FOR PEER REVIEW 8 of 11

FigureFigure 5.5.Genetic Genetic analysis analysis of ORF of 6ORF from 6 ToTLV. from (AToTLV.) PCR and(A) RT-PCR PCR and products RT-PCR from ToTLV-infectedproducts from tomatoToTLV-infected plants. PCRtomato and RT-PCRplants. assays,PCR respectively,and RT-PCR using assays, primers respectively, Be-089F/Be-363R using (1primers and 2). PCRBe-089F/Be-363R and RT-PCR (1 assays, and 2). respectively,PCR and RT-PCR using assays primers, respectively, Be-2147F/Be-363R using primers (3 and Be-2147F/Be-363R 4). (B) Schematic (3 representationand 4). (B) Schematic of ToTLV representation showing the typicalof ToTLV five genesshowing of thethe DNA-A typical componentsfive genes of of Newthe DNA-A World begomovirusescomponents of New (capsid World protein begomoviruses gene (V1), replication-associated(capsid protein gene (V1), protein replication-associated gene (C1), transcriptional protein activatorgene (C1), protein transcriptional gene (C2), activato replicationr protein enhancer gene protein(C2), replication gene (C3), enhancer and the protein C4 protein gene gene (C3), (C4)) and and the theC4 protein additional gene ORF (C4)) 6, whichand the encompasses additional ORF the intergenic6, which encompasses region (IR) andthe theintergenic 50 end region of the V1(IR) gene. and Thethe 5 green′ end lineof the represents V1 gene. the The expected green line amplification represents productthe expected with amplification primer pair Be-089F/Be-363R. product with primer The purplepair Be-089F/Be-363R. line represents the The expected purple amplificationline represents product the expected with primer ampl pairification Be-2147F/Be-363R. product with primer pair Be-2147F/Be-363R. 4. Discussion 4. DiscussionIn this study, genetic and biological analyses showed that the isolate Be6.6H is a new putative speciesIn this of begomovirusstudy, genetic according and biological to the analyses demarcation showed criteria that the established isolate Be6.6H by theis a Internationalnew putative Committeespecies of onbegomovirus Taxonomy ofaccording Viruses [ 1to,3 ]the and demarcat the nameion Tomato criteria twisted established leaf virus by (ToTLV) the International is proposed basedCommittee on the on symptoms Taxonomy observed of Viruses in tomato [1,3] plants.and the ToTLV name was Tomato found twisted in single leaf and virus mixed (ToTLV) infection is inproposed tomato based plants on from the thesymptoms same crop observed field. This in toma virusto exhibitedplants. ToTLV the typical was found genome in single organization and mixed of theinfection DNA-A in componenttomato plants of NW from bipartite the same begomoviruses. crop field. This Phylogenetic virus exhibited analysis the showed typical that genome ToTLV isorganization a member ofof thethe SLCVDNA-A clade, component although of it wasNW notbipartite closely begomoviruses. related to other Phylogenetic members within analysis the groupshowed (Figure that ToTLV3). Recombination is a member evidence of the SLCV was detected clade, although in ToTLV it bywas seven not methodsclosely related implemented to other inmembers the RDP4 within program. the group The (Figure recombination 3). Recombination breakpoints evidence were found was arounddetected the in intergenicToTLV by regionseven 0 andmethods the 5 implementedend of the rep in gene.the RDP4 The program. N-terminal The part recombination of Rep and breakpoints the adjacent were intergenic found around region isthe a recombinationintergenic region hot-spot and the in the 5′ genomeend of the ofbegomoviruses rep gene. The [N-terminal22]. ToYMoV part and of JacYVV Rep and were the suggested adjacent asintergenic putative region parents is involved a recombination in the recombination hot-spot in the event. genome JacYVV of begomoviruses has been found [22]. infecting ToYMoV the weed and J.JacYVV tamnifolia werein suggested Zulia state, as Venezuela putative parents [21] and involved ToTLV wasin the uncovered recombination in the event. same VenezuelanJacYVV has state.been Relationshipsfound infecting between the weed begomoviruses J. tamnifolia in infecting Zulia state, tomato Venezuela and species [21] and of the ToTLV family wasConvolvulaceae uncovered inhave the beensame previously Venezuelan reported state. Relationships in Venezuela [ 23between]. In this be country,gomoviruses the round-year infecting production tomato and and species distribution of the offamily tomato Convolvulaceae seedlings throughout have been major previously tomato-producing reported in states, Venezuela as well as[23]. the In wide this predominance country, the ofround-year polyphagous productionB. tabaci andMiddle distribution East Asia of Minor toma 1to [24 seedlings,25], favors throughout a dynamic major pathosystem tomato-producing leading to diversificationstates, as well as and the evolution wide predominance of begomoviruses. of polyphagous B. tabaci Middle East Asia Minor 1 [24,25], favorsDespite a dynamic several pathosystem attempts basedleading on to RFLP-RCA diversification and and PCR evolution assays, noof begomoviruses. putative cognate DNA-B componentDespite could several be found attempts in association based on withRFLP-RCA the single and component PCR assays, of ToLTV. no putative Hence, thecognate development DNA-B ofcomponent an infectious could clone be wasfound required in association to determine with the the monopartite single component nature of ToTLV.of ToLTV. Agroinoculation Hence, the experimentsdevelopment on of tomato an infectious and N. benthamianaclone was requiredplants confirmed to determine that athe single monopartite DNA component nature of of ToTLV. ToTLV wasAgroinoculation required to establishexperiments the viralon tomato infection. and The N. monopartitebenthamiana plants nature confirmed for an NW that begomovirus a single DNA was demonstratedcomponent of inToTLV 2013 was by two required independent to establish studies the onviral ToLDeV infection. [6, 7The]. To monopartite date, ToLDeV nature has for been an reportedNW begomovirus in Peru, Ecuador, was demonstrated and Brazil in infecting 2013 by only two tomatoes.independent [2,5 studie,6]. Mores on recently,ToLDeV the[6,7]. infectivity To date, ofToLDeV two monopartite has been begomovirusesreported in Peru, from Ecuador, Brazil, Tomatoand Brazil mottle infecting leaf curl virusonly (ToMoLCV)tomatoes. [2,5,6]. and Tomato More leafrecently, curl purplethe infectivity vein virus of two (ToLCPVV), monopartite was begomoviruses also demonstrated from Brazil, [26,27]. Tomato Tomato mottle severe leaf curl leaf virus curl virus(ToMoLCV) (ToSLCV) and has Tomato also been leaf referred curl purple to as vein a monopartite virus (ToLCPVV), NW begomovirus was also indemonstrated Mexico and Central[26,27]. AmericaTomato severe [2]. Although leaf curl ToSLCV virus (ToSLCV) infectivity has has also not been been referred confirmed to as by a inoculationmonopartite of NW infectious begomovirus clones in Mexico and Central America [2]. Although ToSLCV infectivity has not been confirmed by

Viruses 2019, 11, 327 9 of 11 so far, no evidence of the DNA-B component associated with ToSLCV has been found [28,29], even by using a high throughput sequencing strategy [30]. In addition to the typical five ORFs of the NW begomoviruses, ToTLV showed a sixth ORF partially overlapping the 50 end of the CP gene. Interestingly, the AV2/V2 gene from OW begomoviruses is placed in the same region of the genome. This gene is associated with viral movement [31] and can act as an RNA silencing suppressor [32]. The ToTLV ORF 6 had a deduced amino acid sequence of 113 aa, while the size of AV2/V2 proteins range from 113 to 118 aa. However, no significant homology was found between the ORF 6 of ToTLV and any begomovirus protein in the GenBank database. Unlike ToTLV, an AV2-like gene was recently observed in a begomovirus, Sida golden yellow spot virus (SiGYSV), infecting Sida spp. in Brazil [33]. However, SiGYSV is considered to be an OW-like begomovirus as it shares a similar genome size with OW begomovirus (ca. 2800 bp), in addition to the presence of the AV2-like gene [33]. Ho et al. [34] estimated that deletions of more than 100 nt in the proximal promoter region of the AV2/V2 gene may lead to disruption of this gene in NW begomoviruses. In ToTLV, a putative transcript from ORF 6 was suggested by RT-PCR assays (Figure5). Besides, using the criteria to identify a putative TATA box in the CP gene promoter region of geminiviruses [35], we have identified the sequence TATTAT at the position -39 from the potential star codon, which might represent a candidate for this transcription element. However, further studies will be needed to confirm whether this potential transcript is translated and its expression might affect the viral infection. In this work, we have found ToTLV infecting two tomato plants in Venezuela and new surveys in tomato fields will provide insight about the frequency and genetic diversity of this virus in the country. Overall, our results along with previous studies raise new questions about viral movement in plants of NW monopartite begomoviruses in the absence of V2 protein and which mechanisms they use to develop a systemic infection.

Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/11/4/327/s1, Figure S1: (A) Nicotiana benthamiana plant with mosaic symptoms at four weeks after agroinoculation with the ToTLV infectious clone. (B) N. benthamiana plant with no symptoms at four weeks after agroinoculation with the pCAMBIA1300 empty vector as a negative control. Author Contributions: D.T.C. and F.G.-P. carried out sample collection. G.R., M.M., and A.G. performed experiments and data analyses. G.R., A.G., J.M., and C.B. wrote the final draft version of the manuscript. All authors reviewed and approved the manuscript. Funding: The survey was partially supported by the Venezuelan Ministry of Higher Education, Science and Technology, project number FONACIT-G-2000001610. G.R was the recipient of a Move-In Louvain fellowship from the UCLouvain. A.G. held a Chargé de Recherches fellowship from the National Fund for Scientific Research (FNRS). Acknowledgments: We thank Charlotte Lienard, Brigitte Vanpee, and Marie Eve Renard for technical assistance. Conflicts of Interest: The authors declare no conflict of interest.

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